Macrocyclic Compounds Useful as Inhibitors of Histone Deacetylases

ABSTRACT

The present invention provides a novel macrocyclic compound of general Formula (I) having histone deacetylase (HDAC) inhibitory activity, a pharmaceutical composition comprising the compound, and a method useful to treat diseases using the compound.

RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application Ser.No. 61/348,978, filed May 27, 2010, which is incorporated herein byreference in its entirety.

STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH

This invention was made with government support under R01 CA 107098 andR01 CA110246 awarded by National Institute of Health. The government hascertain rights in the invention.

FIELD OF THE INVENTION

The present disclosure relates generally to a series of novelmacrocyclic depsipeptide compounds, having histone deacetylase (HDAC)inhibition properties. In particular, the present disclosure describescompounds that are suitable for use in selectively arresting cellgrowth, inducing terminal differentiation and/or initiating apoptosis ofneoplastic cells thus inhibiting their proliferation. The disclosuredescribes methods of making these novel macrocyclic compounds,pharmaceutical compositions and methods of treatment comprisingselective inhibition HDAC isoforms, resulting in targeted therapies forcancer. Compounds of the invention may also be useful in treatingdiseases driven by HDAC disregulation such as inflammatory diseases,auto immune diseases, allergic diseases and various diseases of thecentral nervous system.

BACKGROUND OF THE INVENTION

Largazole, a cyclic depsipeptide originally isolated from a marinecyanobacterium Symploca sp., has been shown to be an anti-tumor agent(Taori et al. 2008). Largazole specifically targets histone deacetylaseswhose dysfunction is often associated with a variety of human tumors.Largazole has been shown to: (i) display nM GI₅₀ values against avariety of cell lines (eg MDA-MB-231 mammary carcinoma cells, GI₅₀=7.7nM; U2OS fibroblastic osteosarcoma cells, GI₅₀=55 nM; HT29 colon cells,GI₅₀=12 nM; IMR-32 neuroblastoma cells (Taori et al. 2008), GI₅₀=16 nM)(Taori et al. 2008), (ii) display differential activity betweentransformed and non-transformed cells (Nasveschuk et al. 2008; Taori etal. 2008; Ungermannova 2010) and (iii) is structurally simpler andpossibly more tractable synthetically than the other depsipeptides.

Although, the largazole molecule is a proven antitumor agent, there isalways a need for improved structural analogs that lead to improved HDACinhibition properties, toxicity and physiochemical profiles resulting isimproved cancer therapies.

It has been known for years that DMSO and butyrate, two known relativelynonspecific inhibitors of HDACs, can induce certain leukemia cells todifferentiate and suppress neoplastic growth (Sato et al. 1971; Leder etal. 1975).

In recent years, HDACs and histone acetylases (HATs) have become widelyrecognized as key players in regulating transcription (Minucci andPelicci 2006). Acetylation of lysines in the histone H3 and histone H4tails is strongly correlated to chromatin states that are ready fortranscription, or that are part of actively transcribed genomic regions(Allfrey et al. 1964). Acetylation of histones has also been correlatedwith other important cellular functions including chromatin assembly,DNA repair, and recombination.

There are 18 HDAC enzymes in the human genome that can be classifiedinto four classes (Lane and Chabner 2009). Classes I, II and IV allcontain a zinc (Zn²⁺) molecule in their active site (Table 1 adaptedfrom (Lane and Chabner 2009)).

Because of their important role in regulating transcription anddisruptions of their regulation in tumor cells, it has been postulatedthat inhibition o HDAC could be an effective way for cancertherapeutics. Consequently, there has been substantial development ininhibitors of HDAC enzymes (HDACi) as potential anti-cancer drugs(Marks). The clinical relevance of this attention to HDACi is warrantedand has recently been underscored by the introduction of vorinostat(Zolinza™, Merck, also widely known as SAHA=suberoylanilide hydroxamicacid) for the treatment of cutaneous T-cell lymphoma in late 2006 andmore recently Romidepsin (FK228) (Marks).

The catalytic activity of HDAC contains features from both serineprotease and metalloprotease enzymes. On the basis of the crystalstructures of HDAC8 and a bacterial histone deacetylase-like protein(HDLP), the mechanism for the deacetylation reaction has been proposed(Finnin et al. 1999; Somoza et al. 2004; Vannini et al. 2004). There isa deep, tube-like narrow pocket that expands at the bottom and aninternal cavity that borders the pocket (FIG. 1.1). The inside of thetube is comprised of hydrophobic and aromatic residues. The zinc ion issituated at the bottom of the pocket and Zn2+ and His 142, acting as ageneral base, activate the water molecule for nucleophilic attack on thecarbonyl group of the substrate. This would result in a tetrahedralcarbon that is stabilized by the formation of a hydrogen bond with Tyr306 and a general acid His 143 that protonates the lysine leaving group,yielding the acetate and lysine products. Both His 142 and His 143 fitin the Asp 166-His 131 charge-relay system, which is proposed tomodulate the basicity of the His residues (FIG. 1.4) (Finnin et al.1999).

The mode of action of a majority of HDAC inhibitors is to mimic thesubstrate interactions with the deacetylase, thus preventing the entryof the acetylated lysine residue located on the tail of the histoneprotein. All small molecule histone deacetylase inhibitors share threestructural elements that contribute to HDAC inhibition: (1) a surfacerecognition domain which is anchored at the rim of the HDAC's tube-likepocket, (2) a zinc binding site, (3) a linker region that connects thesurface recognition domain to the zinc binding site (Finnin et al.,1999). FIG. 1.5 (adapted form (Newkirk et al. 2009)) shows a generalpharmacophore model of several known HDAC inhibitors.

While SAHA exerts its anti-cancer activity at least in part by themodulation of HDACs in a direct fashion by coordination of the Zn²⁺ ionin the active site of the enzyme by the terminal hydroxamic acid, itdisplays poor selectivity among the 3 classes of HDACs in part due toits structure simplicity (Minucci and Pelicci 2006; Lane and Chabner2009). It has been generally accepted that Class I HDACs are morerelevant to cancer therapy and poor selectivity of HDAC inhibitors areresponsible for chronic toxicities (Minucci and Pelicci 2006; Lane andChabner 2009). In search for more specific Class I HDAC inhibitors hasled to the discovery of a number of natural product depsipeptidesincluding FR901375 (Koho 1991), FK228 (Ueda et al. 1994a),spiruchostatin A (Masuoka et al. 2001), and the very recently isolatedlargazole (Taori et al. 2008) (FIG. 2). These natural products known asDepsipeptides share a common feature in that they all contain an(3S,4E)-3-hydroxy-7-mercapto-4-heptenoic acid side chain (Newkirk et al.2009). A free sulfhydryl (thiol) needs to be exposed in this class ofcompounds to unleash the inhibitory activity of HDAC as the thiolcoordinates the active site Zn²⁺ ion to prevent catalysis.

The Zn²⁺ binding moiety of largazole is inactive unless the thioester isremoved by hydrolysis. It has been demonstrated that largazole-thiol isthe active specie that potently inhibit HDACs (Bowers et al. 2008; Yinget al. 2008b). Thus largazole is most likely a pro-drug that becomesactivated by esterase/lipases upon uptake into cells or conjugated to acarrier/transport protein and reduced to thiol intracellularly. Therehave been significant developments in prodrug design to improvephysicochemical and pharmacokinetic properties of biologically potentlead compounds (Rautio et al. 2008). However, these strategies have notbeen systematically applied to largazole.

SUMMARY OF THE EMBODIMENTS

This invention is related to the field of cancer therapy. In particular,the invention describes novel macrocyclic compounds and pharmaceuticalcompositions comprising them. Further, the invention describes a novelprocess for making and using these compounds. These macrocycliccompounds are HDAC inhibitors and are useful as antiproliferation agentsfor cancer therapy. The methods described herein have identifiedcompounds that have selectivity in targeting HDACs in anti-cancertherapy. These compounds target HDACs whose dysfunction is oftenassociated with a variety of human tumors (Marks and Breslow 2007).

In one aspect, the present invention provides compounds of Formula (I)

Wherein

-   -   “A” is aryl or heteroaryl, optionally substituted with one or        more groups selected from C₁-C₁₀ alkyl, C₃-C₁₀ cycloalkyl,        C₃-C₁₀ heterocycloalkyl, aryl, heteroaryl, halo, hydroxyl, —CN,        —COOH, —CF₃, —OCH₂F, —OR₂₀, —NR₂OR₂₂, —NCOR₂OR₂₂, —CONR₂OR₂₂;    -   Z is —(CH₂)_(n)SR₁₂;    -   R₁ and R₂ are independently H, halo, C₁-C₁₀ alkyl, C₃-C₁₀        cycloalkyl, C₃-C₁₀ heterocycloalkyl,        -   or R₁ and R₂ together,        -   or one of the R₁, R₂ with R₉ form a C₃-C₁₀ cycloalkyl,            C₃-C₁₀ heterocycloalkyl, wherein the C₁-C₁₀ alkyl, C₃-C₁₀            cycloalkyl and C₃-C₁₀ heterocycloalkyl are optionally            substituted with one or more groups selected from C₁-C₁₀            alkyl, C₃-C₁₀ cycloalkyl, C₃-C₁₀ heterocycloalkyl, aryl,            heteroaryl, halo, hydroxyl, —CN, —COOH, —CF₃, —OCH₂F, —OR₂₀,            —NR₂OR₂₂, —NCOR₂OR₂₂, —CONR₂₀R₂₂;    -   R₃ and R₄ are independently H, halo, C₁-C₁₀ alkyl, C₃-C₁₀        cycloalkyl, C₃-C₁₀ heterocycloalkyl,        -   or R₃ and R₄ together,        -   or one of the R₃, R₄ with R₁₀ form a C₃-C₁₀ cycloalkyl,            C₃-C₁₀ heterocycloalkyl,    -   wherein the C₁-C₁₀ alkyl, C₃-C₁₀ cycloalkyl and C₃-C₁₀        heterocycloalkyl are optionally substituted with one or more        groups selected from C₁-C₁₀ alkyl, C₃-C₁₀ cycloalkyl, C₃-C₁₀        heterocycloalkyl, aryl, heteroaryl, halo, hydroxyl, —CN, —COOH,        —CF₃, —OCH₂F, —OR₂₀, —NR₂₀R₂₂, —NCOR₂₀R₂₂, —CONR₂OR₂₂,        —S(O)_(m)R₂₀;    -   R₅ and R₆ are independently H, halo, C₁-C₁₀ alkyl, C₃-C₁₀        cycloalkyl, C₃-C₁₀ heterocycloalkyl,        -   or R₅ and R₆ together,        -   or one of the R₅, R₆ with R₁₁ form a C₃-C₁₀ cycloalkyl,            C₃-C₁₀ heterocycloalkyl,    -   wherein the C₁-C₁₀ alkyl, C₃-C₁₀ cycloalkyl, C₃-C₁₀        heterocycloalkyl are optionally substituted with one or more        groups selected from C₁-C₁₀ alkyl, C₃-C₁₀ cycloalkyl, C₃-C₁₀        heterocycloalkyl, aryl, heteroaryl, halo, hydroxyl, —CN, —COOH,        —CF₃, —OCH₂F, —OR_(D)), —NR₂OR₂₂, —NCOR₂OR₂₂, —CONR₂OR₂₂;    -   R₇ and R₈ are independently H, halo, C₁-C₁₀ alkyl, C₃-C₁₀        cycloalkyl, C₃-C₁₀ heterocycloalkyl,        -   or R₅ and R₆ together form a C₃-C₁₀ cycloalkyl, C₃-C₁₀            heterocycloalkyl, wherein the C₁-C₁₀ alkyl, C₃-C₁₀            cycloalkyl, C₃-C₁₀ heterocycloalkyl are optionally            substituted with one or more groups selected from C₁-C₁₀            alkyl, C₃-C₁₀ cycloalkyl, C₃-C₁₀ heterocycloalkyl, aryl,            heteroaryl, halo, hydroxyl, —CN, —COOH, —CF₃, —OCH₂F, —OR₂₀,            —NR₂OR₂₂, —NCOR₂OR₂₂, —CONR₂₀R₂₂;    -   R₉ is independently H, C₁-C₁₀ alkyl, C₃-C₁₀ cycloalkyl, C₃-C₁₀        heterocycloalkyl,        -   or together with one of R₁, R₂ form a C₃-C₁₀ cycloalkyl,            C₃-C₁₀ heterocycloalkyl,    -   wherein the C₁-C₁₀ alkyl, C₃-C₁₀ cycloalkyl, C₃-C₁₀        heterocycloalkyl are optionally substituted with one or more        groups selected from C₁-C₁₀alkyl, C₃-C₁₀ cycloalkyl, C₃-C₁₀        heterocycloalkyl, aryl, heteroaryl, halo, hydroxyl, —CN, —COOH,        —CF₃, —OCH₂F, —OR₂₀, —NR₂₀R₂₂, —NCOR₂₀R₂₂, —CONR₂₀R₂₂;    -   R₁₀ is independently H, C₁-C₁₀ alkyl, C₃-C₁₀ cycloalkyl, C₃-C₁₀        heterocycloalkyl,        -   or together with one of R₃, R₄ form a C₃-C₁₀ cycloalkyl,            C₃-C₁₀ heterocycloalkyl,    -   wherein the C₁-C₁₀ alkyl, C₃-C₁₀ cycloalkyl, C₃-C₁₀        heterocycloalkyl are optionally substituted with one or more        groups selected from C₁-C₁₀ alkyl, C₃-C₁₀ cycloalkyl, C₃-C₁₀        heterocycloalkyl, aryl, heteroaryl, halo, hydroxyl, —CN, —COOH,        —CF₃, —OCH₂F, —OR₂₀, —NR₂OR₂₂, —NCOR₂OR₂₂, —CONR₂₀R₂₂;    -   R₁₁ is independently H, C₁-C₁₀ alkyl, C₃-C₁₀ cycloalkyl, C₃-C₁₀        heterocycloalkyl,        -   or together with one of R₅, R₆ form a C₃-C₈cycloalkyl, C₃-C₈            heterocycloalkyl,    -   wherein the C₁-C₁₀alkyl, C₃-C₁₀ cycloalkyl, C₃-C₁₀        heterocycloalkyl are optionally substituted with one or more        groups selected from C₁-C₈ alkyl, C₃-C₈ cycloalkyl, C₃-C₈        heterocycloalkyl, aryl, heteroaryl, halo, hydroxyl, —CN, —COOH,        —CF₃, —OCH₂F, —OR₂₀, —NR₂₀R₂₂, —NCOR₂₀R₂₂, —CONR₂₀R₂₂;    -   R₁₂ is independently H, C₁-C₁₀ alkyl, —COR₂₀, —CONR₂OR₂₂, —OR₂₀,        —COOR₂₀, —COCR₂OR₂₂NR₂OR₂₂, —SR₂₀, —P(O)(OR₂₄)₂;    -   R₂₀ and R₂₂ are independently H, C₁-C₁₀ alkyl, C₃-C₁₀        cycloalkyl, C₃-C₁₀ heterocycloalkyl, aryl or heteroaryl;    -   R₂₄ is independently H, C₁-C₁₀ alkyl, C₃-C₁₀ cycloalkyl, C₃-C₁₀        heterocycloalkyl, Na, K or Ca;    -   n=1-6;    -   m=1 or 2;    -   or a pharmaceutically acceptable salt, solvate, prodrug or        stereoisomer thereof.

In another aspect, the present invention provides compounds of Formula(II)

Wherein

-   -   L and Q are independently S, O, N, or CR₂₆    -   R₂₆ is independently H, halo, C₁-C₁₀ alkyl, C₃-C₁₀ cycloalkyl,        C₃-C₁₀ heterocycloalkyl, aryl, heteroaryl, wherein the C₁-C₁₀        alkyl, C₃-C₁₀ cycloalkyl, C₃-C₁₀ heterocycloalkyl, aryl and        heteroaryl are optionally substituted with one or more groups        selected from C₁-C₁₀ alkyl, C₃-C₁₀ cycloalkyl, C₃-C₁₀        heterocycloalkyl, aryl, heteroaryl, halo, hydroxyl, —CN, —COOH,        —CF₃, —OCH₂F, —OR₂₀, —NR₂₀R₂₂, —NCOR₂₀R₂₂, —CONR₂OR₂₂;    -   R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈, R₉, R₁₀, R₁₁ and Z are as        described above;    -   or a pharmaceutically acceptable salt, solvate, prodrug or        stereoisomer thereof.

In yet another aspect, the present invention provides compounds ofFormula (III)

Wherein

-   -   L, Q and Y are independently S, O, N, or CR₂₆;    -   R₂₆ is independently H, halo, C₁-C₁₀ alkyl, C₃-C₁₀ cycloalkyl,        C₃-C₁₀ heterocycloalkyl, aryl, heteroaryl, wherein the C₁-C₁₀        alkyl, C₃-C₁₀cycloalkyl, C₃-C₁₀ heterocycloalkyl, aryl and        heteroaryl are optionally substituted with one or more groups        selected from C₁-C₁₀ alkyl, C₃-C₁₀ cycloalkyl, C₃-C₁₀        heterocycloalkyl, aryl, heteroaryl, halo, hydroxyl, —CN, —COOH,        —CF₃, —OCH₂F, —OR₂₀, —NR₂OR₂₂, —NCOR₂₀R₂₂, —CONR₂OR₂₂;    -   R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈, R₉, R₁₀, R₁₁ and Z are as        described above;    -   or a pharmaceutically acceptable salt, solvate, prodrug or        stereoisomer thereof.

In yet another aspect, the present invention provides compound ofFormula (IV)

or a pharmaceutically acceptable salt, solvate, prodrug or stereoisomerthereof.

In yet another aspect, the present invention provides compound ofFormula (V)

or a pharmaceutically acceptable salt, solvate, prodrug or stereoisomerthereof.

In yet another aspect, the present invention provides compound ofFormula (VI)

or a pharmaceutically acceptable salt, solvate, prodrug or stereoisomerthereof.

In yet another aspect, the present invention provides compound ofFormula (VII)

or a pharmaceutically acceptable salt, solvate, prodrug or stereoisomerthereof.

In yet another aspect, the present invention provides compound ofFormula (VIII)

or a pharmaceutically acceptable salt, solvate, prodrug or stereoisomerthereof.

In yet another aspect, the present invention provides compound ofFormula (IX)

or a pharmaceutically acceptable salt, solvate, prodrug or stereoisomerthereof.

In yet another aspect, the present invention provides compound ofFormula (X)

Wherein A, R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈, R₉, R₁₀, R₁₁ and n are asdescribed above;or a pharmaceutically acceptable salt, solvate, prodrug or stereoisomerthereof.

In yet another aspect, the present invention provides pharmaceuticalcompositions of compounds or pharmaceutically acceptable salts of one ormore compounds described herein and a pharmaceutically acceptablecarrier.

In yet another aspect, the present invention provides methods oftreating diseases mediated by HDAC enzymes, comprising administering toa subject in need thereof a therapeutically effective amount of one ormore compounds described herein. Other methods involve co-therapies byadministering one or more compounds of the present invention with otheranti-cancer agents.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a potential role of HAT and HDAC in transcriptionalregulation.

-   -   A) Histone modification by HAT and HDAC.    -   B) Regulation of gene expression switches by co-activator or        co-repressor complex. Figure and description adapted from (Kim        et al. 2003).

FIG. 2 presents several embodiments of HDLP-TSA complexes.

-   -   A) Space-filling representation of TSA in the active-site        pocket. The hydroxamic acid group, most of the aliphatic chain        and part of the dimethylamino-phenyl group of TSA are buried        (60% of TSA's surface area).    -   B) Schematic representation of HDLP-TSA interactions. Both        panels and partial description was adapted from (Finnin et al.        1999).

FIG. 3 presents a proposed mechanism of action of zinc-dependent HDACs.

FIG. 4 illustrates one embodiment of a HDACi pharmacophore. Cap regionon top; linker on the bottom with zinc-binding moiety. Figure anddescription adapted from (Newkirk et al. 2009).

FIG. 5. Natural product depsipeptide HDAC inhibitors. The keythiol-containing domain is shown.

FIG. 6 depicts a possible activation of largazole and FK228 to carry outHDAC inhibition. Largazole is a prodrug that upon hydrolysis isconverted to the corresponding thiol, which deactivates HDAC bychelating zinc away from the active site of the enzyme. In a similarmanner, reduction of the disulfide bond in FK228 liberates thiol thatpotently inhibits HDACs.

FIG. 7 provides the structures of largazole, and several structuralanalogues of largazole. Example 1 is also stated as CGN 552.

FIG. 8 presents exemplary data showing that largazole, its selectedanalogues, and SAHA promote H3 hyperacetylation in the HCT 116 cancercell line.

a) Largazole (L) and SAHA inhibit H3 deacetylation in a time-dependentmanner. HCT116 cells were treated with 10 nM largazole or 200 nM SAHAfor the times indicated in the panel. Cell extracts were separated viaSDS-PAGE electrophoresis and the resulting bands were detected utilizinganti-acetyl-H3 antibodies. DMSO-treated cells serve as a negativecontrol, while the expression of GAPDH was used to show equal loading.b) HCT116 cells respond to largazole, its selective analogues, and SAHAin a dose-dependent fashion. Cells were treated with the indicatedcompound for 8 hours with concentrations ranging from 1/μM to 100 nM andimmunoblotted with anti-acetylhistone H3 antibody. CGN-722, CGN-552 andCGN-596 are slightly more potent deacetylase inhibitors than largazole(L) and SAHA. Conversion of thioester into ketone, (CGN-363), rendersthe compound inactive with respect to HDAC inhibition.

FIG. 9 presents DNA microarray data showing that SAHA, Largazole and itsselected analog example 1 cause distinct changes in gene expressionprofiles in the HCT 116 cancer cell line. a) hierarchical clustering andheatmaps of changes in gene expression profiles in the HCT116 cells withindicated treatment. b) Venn diagrams showing unique and shared genesets whose expression levels changed by more than 2 fold upon exposureto the indicated treatment at 6 hr and 24 hr.

Table 1 presents classification of HDAC isoforms. Class I consists ofHDAC 1, 2, 3, and 8. Class II consists of HDAC 4, 5, 6, 7, 9, 10.

Table 2 presents exemplary data showing an effect of largazole,largazole analogs along with example compounds of present invention oncancer cell growth inhibition.

Comparative analysis of the cytotoxic effect of largazole in humancancer cell lines HCT116, SW480 and MDA-MB231. HME were used as control.8,000 cells were plated in 96-well plates where rows 1 and 10 weretreated with DMSO, row 2 had no cells to establish background levels,rows 3-9 contained decreasing concentrations of compound. Cells wereincubated with largazole for 48 hours, followed by staining with crystalviolet dye. The absorbance at 588 nM was measured using a Tecan Sail reII plate reader. Experiments were performed in replicates of six, andconcentration-response curves were generated by nonlinear least squareregression analysis of the data using GraphPad Prism (San Diego,Calif.). The growth inhibition (GI₅₀) for each compound was defined as aconcentration of drug leading to a 50% reduction in A588 compared withcontrols

Table 3 shows a summary of the number of genes whose expression levelschanged by 2 fold upon treatment with indicated chemicals in comparisonto DMSO. Expression values files for each sample were generated usingthe Robust Multichip Average (RMA) algorithm. Differential expressionwas determined using the R software package limma to generate linearmodels and empirical Bayesian statistics. Genes were considereddifferentially expressed if the P value, adjusted for multiple testingusing the Benjamini and Hochberg method, was <5% and the log-2 foldchange was ≦1 or ≧−1.

DETAILED DESCRIPTION

The following description is merely exemplary in nature and is notintended to limit the present disclosure, application, or uses.

DEFINITIONS

The term “substitute for” as used herein, refers to switching theadministration of a first compound or drug to a subject for a secondcompound or drug to the subject. For example, a Kratom extract may besubstituted for an addictive compound such that a subject will beadministered the Kratom extract instead of the addictive compound.

The term “at risk for” as used herein, refers to a medical condition orset of medical conditions exhibited by a patient which may predisposethe patient to a particular disease or affliction. For example, theseconditions may result from influences that include, but are not limitedto, behavioral, emotional, chemical, biochemical, or environmentalinfluences.

The term “effective amount” as used herein, refers to a particularamount of a pharmaceutical composition comprising a therapeutic agentthat achieves a clinically beneficial result (i.e., for example, areduction of symptoms). Toxicity and therapeutic efficacy of suchcompositions can be determined by standard pharmaceutical procedures incell cultures or experimental animals, e.g., for determining the LD₅₀(the dose lethal to 50% of the population) and the ED₅₀ (the dosetherapeutically effective in 50% of the population). The dose ratiobetween toxic and therapeutic effects is the therapeutic index, and itcan be expressed as the ratio LD₅₀/ED₅₀. Compounds that exhibit largetherapeutic indices are preferred. The data obtained from these cellculture assays and additional animal studies can be used in formulatinga range of dosage for human use. The dosage of such compounds liespreferably within a range of circulating concentrations that include theED₅₀ with little or no toxicity. The dosage varies within this rangedepending upon the dosage form employed, sensitivity of the patient, andthe route of administration.

The term “symptom”, as used herein, refers to any subjective orobjective evidence of disease or physical disturbance observed by thepatient. For example, subjective evidence is usually based upon patientself-reporting and may include, but is not limited to, pain, headache,visual disturbances, nausea and/or vomiting. Alternatively, objectiveevidence is usually a result of medical testing including, but notlimited to, body temperature, complete blood count, lipid panels,thyroid panels, blood pressure, heart rate, electrocardiogram, tissuebody imaging scans and other medical testing results.

The term “disease”, as used herein, refers to any impairment of thenormal state of the living animal or one of its parts that interrupts ormodifies the performance of the vital functions. Typically manifested bydistinguishing signs and symptoms, it is usually a response to: i)environmental factors (as malnutrition, industrial hazards, or climate);ii) specific infective agents (as worms, bacteria, or viruses); iii)inherent defects of the organism (as genetic anomalies); and/or iv)combinations of these factors

The terms “reduce,” “inhibit,” “diminish,” “suppress,” “decrease,”“prevent” and grammatical equivalents (including “lower,” “smaller,”etc.) when in reference to the expression of any symptom in an untreatedsubject relative to a treated subject, mean that the quantity and/ormagnitude of the symptoms in the treated subject is lower than in theuntreated subject by any amount that is recognized as clinicallyrelevant by any medically trained personnel. In one embodiment, thequantity and/or magnitude of the symptoms in the treated subject is atleast 10% lower than, at least 25% lower than, at least 50% lower than,at least 75% lower than, and/or at least 90% lower than the quantityand/or magnitude of the symptoms in the untreated subject.

The term “inhibitory compound” as used herein, refers to any compoundcapable of interacting with (i.e., for example, attaching, binding etc.)to a binding partner under conditions such that the binding partnerbecomes unresponsive to its natural ligands. Inhibitory compounds mayinclude, but are not limited to, small organic molecules, antibodies,and proteins/peptides.

The term “attached” as used herein, refers to any interaction between amedium (or carrier) and a drug. Attachment may be reversible orirreversible. Such attachment includes, but is not limited to, covalentbonding, ionic bonding, Van der Waals forces or friction, and the like.A drug is attached to a medium (or carrier) if it is impregnated,incorporated, coated, in suspension with, in solution with, mixed with,etc.

The term “drug” or “compound” as used herein, refers to anypharmacologically active substance capable of being administered whichachieves a desired effect. Drugs or compounds can be synthetic ornaturally occurring, non-peptide, proteins or peptides, oligonucleotidesor nucleotides, polysaccharides or sugars.

The term “administered” or “administering”, as used herein, refers toany method of providing a composition to a patient such that thecomposition has its intended effect on the patient. An exemplary methodof administering is by a direct mechanism such as, local tissueadministration (i.e., for example, extravascular placement), oralingestion, transdermal patch, topical, inhalation, suppository etc.

The term “patient”, as used herein, is a human or animal and need not behospitalized. For example, out-patients, persons in nursing homes are“patients.” A patient may comprise any age of a human or non-humananimal and therefore includes both adult and juveniles (i.e., children).It is not intended that the term “patient” connote a need for medicaltreatment, therefore, a patient may voluntarily or involuntarily be partof experimentation whether clinical or in support of basic sciencestudies.

The term “subject” as used herein refers to a vertebrate, preferably amammal, more preferably a primate, still more preferably a human.Mammals include, without limitation, humans, primates, wild animals,feral animals, farm animals, sports animals, and pets.

The term “affinity” as used herein, refers to any attractive forcebetween substances or particles that causes them to enter into andremain in chemical combination. For example, an inhibitor compound thathas a high affinity for a receptor will provide greater efficacy inpreventing the receptor from interacting with its natural ligands, thanan inhibitor with a low affinity.

The term “derived from” as used herein, refers to the source of acompound or sequence. In one respect, a compound or sequence may bederived from an organism or particular species. In another respect, acompound or sequence may be derived from a larger complex or sequence.

The term “test compound” as used herein, refers to any compound ormolecule considered a candidate as an inhibitory compound.

The term “protein” as used herein, refers to any of numerous naturallyoccurring extremely complex substances (as an enzyme or antibody) thatconsist of amino acid residues joined by peptide bonds, contain theelements carbon, hydrogen, nitrogen, oxygen, usually sulfur. In general,a protein comprises amino acids having an order of magnitude within thehundreds.

The term “peptide” as used herein, refers to any of various amides thatare derived from two or more amino acids by combination of the aminogroup of one acid with the carboxyl group of another and are usuallyobtained by partial hydrolysis of proteins. In general, a peptidecomprises amino acids having an order of magnitude with the tens.

The term “pharmaceutically” or “pharmacologically acceptable”, as usedherein, refer to molecular entities and compositions that do not produceadverse, allergic, or other untoward reactions when administered to ananimal or a human.

The term, “pharmaceutically acceptable carrier”, as used herein,includes any and all solvents, or a dispersion medium including, but notlimited to, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyethylene glycol, and the like), suitable mixturesthereof, and vegetable oils, coatings, isotonic and absorption delayingagents, liposome, commercially available cleansers, and the like.Supplementary bioactive ingredients also can be incorporated into suchcarriers.

The term, “purified” or “isolated”, as used herein, may refer to apeptide composition that has been subjected to treatment (i.e., forexample, fractionation) to remove various other components, and whichcomposition substantially retains its expressed biological activity.

The term “sample” as used herein is used in its broadest sense andincludes environmental and biological samples. Environmental samplesinclude material from the environment such as soil and water. Biologicalsamples may be animal, including, human, fluid (e.g., blood, plasma andserum), solid (e.g., stool), tissue, liquid foods (e.g., milk), andsolid foods (e.g., vegetables). For example, a pulmonary sample may becollected by bronchoalveolar lavage (BAL) which comprises fluid andcells derived from lung tissues. A biological sample may comprise acell, tissue extract, body fluid, chromosomes or extrachromosomalelements isolated from a cell, genomic DNA (in solution or bound to asolid support such as for Southern blot analysis), RNA (in solution orbound to a solid support such as for Northern blot analysis), cDNA (insolution or bound to a solid support) and the like.

The term “biologically active” refers to any molecule having structural,regulatory or biochemical functions. For example, biological activitymay be determined, for example, by restoration of wild-type growth incells lacking protein activity. Cells lacking protein activity may beproduced by many methods (i.e., for example, point mutation andframe-shift mutation). Complementation is achieved by transfecting cellswhich lack protein activity with an expression vector which expressesthe protein, a derivative thereof, or a portion thereof.

The term “label” or “detectable label” are used herein, to refer to anycomposition detectable by spectroscopic, photochemical, biochemical,immunochemical, electrical, optical or chemical means. Such labelsinclude biotin for staining with labeled streptavidin conjugate,magnetic beads (e.g., Dynabeads®), fluorescent dyes (e.g., fluorescein,texas red, rhodamine, green fluorescent protein, and the like),radiolabels (e.g., ³H, ¹²⁵I, ³⁵S, ¹⁴C, or ³²P), enzymes (e.g., horseradish peroxidase, alkaline phosphatase and others commonly used in anELISA), and calorimetric labels such as colloidal gold or colored glassor plastic (e.g., polystyrene, polypropylene, latex, etc.) beads.Patents teaching the use of such labels include, but are not limited to,U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437;4,275,149; and 4,366,241 (all herein incorporated by reference). Thelabels contemplated in the present invention may be detected by manymethods. For example, radiolabels may be detected using photographicfilm or scintillation counters, fluorescent markers may be detectedusing a photodetector to detect emitted light. Enzymatic labels aretypically detected by providing the enzyme with a substrate anddetecting, the reaction product produced by the action of the enzyme onthe substrate, and calorimetric labels are detected by simplyvisualizing the colored label.

The term “conjugate”, as used herein, refers to any compound that hasbeen formed by the joining of two or more moieties.

A “moiety” or “group” is any type of molecular arrangement designated byformula, chemical name, or structure. Within the context of certainembodiments, a conjugate is said to comprise one or more moieties orchemical groups. This means that the formula of the moiety issubstituted at some place in order to be joined and be a part of themolecular arrangement of the conjugate. Although moieties may bedirectly covalently joined, it is not intended that the joining of twoor more moieties must be directly to each other. A linking group,crosslinking group, or joining group refers any molecular arrangementthat will connect the moieties by covalent bonds such as, but are notlimited to, one or more amide group(s), may join the moieties.Additionally, although the conjugate may be unsubstituted, the conjugatemay have a variety of additional substituents connected to the linkinggroups and/or connected to the moieties.

A “polymer” or “polymer group” means a chemical species or group made upof repeatedly linked moieties. Within certain embodiments, it ispreferred that the number repeating moieties is three or more or greaterthan 10. The linked moieties may be identical in structure or may havevariation of moiety structure. A “monomeric polymer” or “homopolymer” isa polymer that contains the same repeating, asymmetric subunit. A“copolymer” is a polymer that is derived from two or more types ofmonomeric species, i.e. two or more different chemical asymmetricsubunits. “Block copolymers” are polymers comprised of two or morespecies of polymer subunits linked by covalent bonds.

The term “substituted”, as used herein, means at least one hydrogen atomof a molecular arrangement is replaced with a substituent. In the caseof an oxo substituent (“═O”), two hydrogen atoms are replaced. Whensubstituted, one or more of the groups below are “substituents.”Substituents include, but are not limited to, halogen, hydroxy, oxo,cyano, nitro, amino, alkylamino, dialkylamino, alkyl, alkoxy, alkylthio,haloalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, heterocycle,and heterocyclealkyl, as well as, —NRaRb, —NRaC(═O)Rb, —NRaC(═O)NRaNRb,—NRaC(═O)ORb, —NRaSO₂Rb, —C(═O)Ra, —C(═O)ORa, —C(═O)NRaRb, —OC(═O)NRaRb,—OR, —SR, —SORo, —S(═O)aR, —OS(═O)₂Ra and —S(═O)ORa. In addition, theabove substituents may be further substituted with one or more of theabove substituents, such that the substituent comprises a substitutedalky, substituted aryl, substituted arylalkyl, substituted heterocycle,or substituted heterocyclealkyl. Ra and Rb in this context may be thesame or different and, independently, hydrogen, alkyl, haloalkyl,substituted alkyl, aryl, substituted aryl, arylalkyl, substitutedarylalkyl, heterocycle, substituted heterocycle, heterocyclealkyl orsubstituted heterocyclealkyl.

The term “unsubstituted”, as used herein, refers to any compound thatdoes not contain extra substituents attached to the compound. Anunsubstituted compound refers to the chemical makeup of the compoundwithout extra substituents, e.g., the compound does not containprotecting group(s). For example, unsubstituted proline is a prolineamino acid even though the amino group of proline may be considereddisubstituted with alkyl groups.

The term “alkyl”, as used herein, means any straight chain or branched,non-cyclic or cyclic, unsaturated or saturated aliphatic hydrocarboncontaining from 1 to 10 carbon atoms, while the term “lower alkyl” hasthe same meaning as alkyl but contains from 1 to 6 carbon atoms. Theterm “higher alkyl” has the same meaning as alkyl but contains from 2 to10 carbon atoms. Representative saturated straight chain alkyls include,but are not limited to, methyl, ethyl, n-propyl, n-butyl, n-pentyl,n-hexyl, n-septyl, n-octyl, n-nonyl, and the like; while saturatedbranched alkyls include, but are not limited to, isopropyl, sec-butyl,isobutyl, tert-butyl, isopentyl, and the like. Cyclic alkyls may beobtained by joining two alkyl groups bound to the same atom or byjoining two alkyl groups each bound to adjoining atoms. Representativesaturated cyclic alkyls include, but are not limited to, cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, and the like; while unsaturatedcyclic alkyls include, but are not limited to, cyclopentenyl andcyclohexenyl, and the like. Cyclic alkyls are also referred to herein asa “homocycles” or “homocyclic rings.” Unsaturated alkyls contain atleast one double or triple bond between adjacent carbon atoms (referredto as an “alkenyl” or “alkynyl”, respectively). Representative straightchain and branched alkenyls include, but are not limited to, ethylenyl,propylenyl, 1-butenyl, 2-butenyl, isobutylenyl, 1-pentenyl, 2-pentenyl,3-methyl-1-butenyl, 2-methyl-2-butenyl, 2,3-dimethyl-2-butenyl, and thelike; while representative straight chain and branched alkynyls include,but are not limited to, acetylenyl, propynyl, 1-butynyl, 2-butynyl,1-pentynyl, 2-pentynyl, 3-methyl-1-butynyl, and the like.

The term “aryl”, as used herein, means any aromatic carbocyclic moietysuch as, but not limited to, phenyl or naphthyl.

The term “arylalkyl”, or “aralkyl” as used herein, means any alkylhaving at least one alkyl hydrogen atoms replaced with an aryl moiety,such as benzyl, but not limited to, —(CH₂)₂-phenyl, —(CH₂)₃-phenyl,—CH(phenyl)₂, and the like.

The term “halogen”, as used herein, refers to any fluoro, chloro, bromo,or iodo moiety.

The term “haloalkyl”, as used herein, refers to any alkyl having atleast one hydrogen atom replaced with halogen, such as trifluoromethyl,and the like.

The term “heteroaryl”, as used herein, refers to any aromaticheterocycle ring of 5 to 10 members and having at least one heteroatomselected from nitrogen, oxygen and sulfur, and containing at least 1carbon atom, including, but not limited to, both mono and bicyclic ringsystems. Representative heteroaryls include, but are not limited to,furyl, benzofuranyl, thiophenyl, benzothiophenyl, pyrrolyl, indolyl,isoindolyl, azaindolyl, pyridyl, quinolinyl, isoquinolinyl, oxazolyl,isooxazolyl, benzoxazolyl, pyrazolyl, imidazolyl, benzimidazolyl,thiazolyl, benzothiazolyl, isothiazolyl, pyridazinyl, pyrimidinyl,pyrazinyl, triazinyl, cinnolinyl, phthalazinyl, or quinazolinyl.

The term “heteroarylalkyl”, as used herein, means any alkyl having atleast one alkyl hydrogen atom replaced with a heteroaryl moiety, such as—CHpyridinyl, CH₂pyrimidinyl, and the like.

The term “heterocycle” or “heterocyclic ring”, as used herein, means any4- to 7-membered monocyclic, or 7- to 10-membered bicyclic, heterocyclicring which is either saturated, unsaturated, or aromatic, and whichcontains from 1 to 4 heteroatoms independently selected from nitrogen,oxygen and sulfur, and wherein the nitrogen and sulfur heteroatoms maybe optionally oxidized, and the nitrogen heteroatom may be optionallyquaternized, including bicyclic rings in which any of the aboveheterocycles are fused to a benzene ring. The heterocycle may beattached via any heteroatom or carbon atom. Heterocycles may includeheteroaryls exemplified by those defined above. Thus, in addition to theheteroaryls listed above, heterocycles may also include, but are notlimited to, morpholinyl, pyrrolidinonyl, pyrrolidinyl, piperidinyl,hydantoinyl, valerolactamyl, oxiranyl, oxetanyl, tetrahydrofuranyl,tetrahydropyranyl, tetrahydropyridinyl, tetrahydroprimidinyl,tetrahydrothiophenyl, tetrahydrothiopyranyl, tetrahydropyrimidinyl,tetrahydrothiophenyl, tetrahydrothiopyranyl, and the like.

The term “heterocycloalkyl”, as used herein, means any alkyl having atleast one alkyl hydrogen atom replaced with a heterocycle, such as—CH₂-morpholinyl, and the like.

The term “homocycle” or “cycloalkyl”, as used herein, means anysaturated or unsaturated (but not aromatic) carbocyclic ring containingfrom 3-7 carbon atoms, such as, but not limited to, cyclopropane,cyclobutane, cyclopentane, cyclohexane, cycloheptane, cyclohexene, andthe like.

The term “alkylamino”, as used herein, means at least one alkyl moietyattached through a nitrogen bridge (i.e., —N-(alkyl)N, such as adialkylamino)) including, but not limited to, methylamino, ethylamino,dimethylamino, diethylamino, and the like.

The term “alkyloxy” or “alkoxy”, as used herein, means any alkyl moietyattached through an oxygen bridge (i.e., —O-alkyl) such as, but notlimited to, methoxy, ethoxy, and the like.

The term “alkylthio”, as used herein, means any alkyl moiety attachedthrough a sulfur bridge (i.e., —S— alkyl) such as, but not limited to,methylthio, ethylthio, and the like

The term “alkenyl” means an unbranched or branched hydrocarbon chainhaving one or more double bonds therein. The double bond of an alkenylgroup can be unconjugated or conjugated to another unsaturated group.Suitable alkenyl groups include, but are not limited to vinyl, allyl,butenyl, pentenyl, hexenyl, butadienyl, pentadienyl, hexadienyl,2-ethylhexenyl, 2-propyl-2-butenyl,4(2-methyl-3-butene)-pentenyl. Analkenyl group can be unsubstituted or substituted with one or twosuitable substituents.

The term “alkynyl” means unbranched or branched hydrocarbon chain havingone or more triple bonds therein. The triple bond of an alkynyl groupcan be unconjugated or conjugated to another unsaturated group. Suitablealkynyl groups include, but are not limited to ethynyl, propynyl,butynyl, pentynyl, hexynyl, methylpropynyl, 4-methyl-1-butynyl,4-propyl-2-pentynyl-, and 4-butyl-2-hexynyl. Analkynyl group can beunsubstituted or substituted with one or two suitable substituents

The term “salts”, as used herein, refers to any salt that complexes withidentified compounds contained herein. Examples of such salts include,but are not limited to, acid addition salts formed with inorganic acids(e.g. hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoricacid, nitric acid, and the like), and salts formed with organic acidssuch as, but not limited to, acetic acid, oxalic acid, tartaric acid,succinic acid, malic acid, fumaric acid, maleic acid, ascorbic acid,benzoic acid, tannic acid, pamoic acid, alginic acid, polyglutamic,acid, naphthalene sulfonic acid, naphthalene disulfonic acid, andpolygalacturonic acid. Salt compounds can also be administered aspharmaceutically acceptable quaternary salts known by a person skilledin the art, which specifically include the quaternary ammonium salts ofthe formula —NR,R′,R″+Z—, wherein R, R′, R″ is independently hydrogen,alkyl, or benzyl, and Z is a counter ion, including, but not limited to,chloride, bromide, iodide, alkoxide, toluenesulfonate, methylsulfonate,sulfonate, phosphate, or carboxylate (such as benzoate, succinate,acetate, glycolate, maleate, malate, fumarate, citrate, tartrate,ascorbate, cinnamoate, mandeloate, and diphenylacetate). Salt compoundscan also be administered as pharmaceutically acceptable pyridine cationsalts having a substituted or unsubstituted partial formula: wherein Zis a counter ion, including, but not limited to, chloride, bromide,iodide, alkoxide, toluenesulfonate, methylsulfonate, sulfonate,phosphate, or carboxylate (such as benzoate, succinate, acetate,glycolate, maleate, malate, fumarate, citrate, tartrate, ascorbate,cinnamoate, mandeloate, and diphenylacetate).

As used herein, the term “prodrug” refers to a derivative of a compoundthat can hydrolyze, oxidize, or otherwise react under biologicalconditions (in vitro or in vivo) to provide a compound of the invention.Prodrugs may only become active upon some reaction under biologicalconditions, but they may have activity in their unreacted forms.Examples of prodrugs contemplated herein include, without limitation,analogs or derivatives of compounds of the invention, and/or their saltswhen salt formation is possible, but in particular, derivatives of zincbinding thiol moiety. Examples of prodrug moieties include substitutedand unsubstituted, branched or unbranched lower alkyl ester moieties,(e.g., propionic acid esters), lower alkenyl esters, di-loweralkyl-amino lower-alkyl esters (e.g., dimethylaminoethyl ester),acylamino lower alkyl esters (e.g., acetyloxymethyl ester), acyloxylower alkyl esters (e.g., pivaloyloxymethyl ester), aryl esters (phenylester), aryl-lower alkyl esters (e.g., benzyl ester), heteroaryl esters(nicotinate ester), substituted (e.g., with methyl, halo, or methoxysubstituents) aryl and aryl-lower alkyl esters, amides, lower-alkylamides, di-lower alkyl amides, and hydroxy amides. Naturally occurringamino acid esters or their enantiomers, dipeptide esters, phosphateesters, methoxyphosphate esters, disulfides and disulfide dimers.Prodrugs and their uses are well known in the art (see, e.g., Berge etal. 1977). Prodrugs can typically be prepared using well-known methods,such as those described in Burger's Medicinal Chemistry and DrugDiscovery (Manfred E. Wolff ed. 1995) and (Rautio, 2008).

As used herein, “reactive groups” refer to nucleophiles, electrophiles,or radically active groups, i.e., groups that react in the presence ofradicals. A nucleophile is a moiety that forms a chemical bond to itsreaction partner (the electrophile) by donating both bonding electrons.Electrophiles accept these electrons. Nucleophiles may take part innucleophilic substitution, whereby a nucleophile becomes attracted to afull or partial positive charge on an element and displaces the group itis bonded to. Alternatively nucleophiles may take part in substitutionof carbonyl group. Carboxylic acids are often made electrophilic bycreating succinyl esters and reacting these esters with aminoalkyls toform amides. Other common nucleophilic groups are thiolalkyls,hydroxylalkys, primary and secondary amines, and carbon nucleophilessuch as enols and alkyl metal complexes. Other preferred methods ofligating proteins, oligosaccharides and cells using reactive groups aredisclosed in (Lemieux and Bertozzi 1998), incorporated herein byreference. In yet another preferred method, one provides reactive groupsfor the Staudinger ligation, i.e., “click chemistry” with an azidecomprising moiety and alkynyl reactive groups to form triazoles. Michealadditions of a carbon nucleophile enolate with an electrophiliccarbonyl, or the Schiffbase formation of a nucleophilic primary orsecondary amine with an aldehyde or ketone may also be utilized. Othermethods of bioconjugation are provided in (Hang and Bertozzi 2001) and(Kiick et al. 2002), both of which are incorporated by reference.

The term “biocompatible”, as used herein, refers to any material thatdoes not illicit a substantial detrimental response in the host. Thereis always concern, when a foreign object is introduced into a livingbody, that the object will induce an immune reaction, such as aninflammatory response that will have negative effects on the host. Inthe context of this invention, biocompatibility is evaluated accordingto the application for which it was designed: for example; a bandage isregarded a biocompatible with the skin, whereas an implanted medicaldevice is regarded as biocompatible with the internal tissues of thebody. Preferably, biocompatible materials include, but are not limitedto, biodegradable and biostable materials. A substantial detrimentalresponse has not occurred if an implant comprising the material is inclose association to its implant site within the host animal and theresponse is better than a tissue response recognized and established assuitable from a materials provided in an ASTM. ASTM subcommittee F04.16on Biocompatibility Test Methods has developed biocompatibilitystandards for medical and surgical materials and devices. For example,materials that are to be used in contact with the blood stream must becomposed of materials that meet hemocompatibilty standards. One of thesetests is for damage to red blood cells, which can result in hemolysisthat is, rupturing of the cells, as described in F 756 Practice forAssessment of Hemolytic Properties of Materials, incorporated herein byreference.

As used herein, a “bioactive substance” refers to any of a variety ofchemical moieties and that binds with a biomolecule such as, but notlimited to, peptides, proteins, enzymes, receptors, substrates, lipids,antibodies, antigens, and nucleic acids. In certain preferredembodiments, the bioactive substance is a biomolecule but it notintended that the bioactive substance be limited to biomolecules. Inother preferred embodiments, the bioactive substances providehydrophobic, hydrophilic or electrostatic interactions, such aspolycarboxylic acids that are anionic at physiological pH. In otherpreferred embodiment, the alkaline growth factors (with isoelectricpoint above 7) are retained via favorable electrostatic interactions bythe polycarboxylates, and subsequently released in a controlled andsustained manner.

“Cancer” is a term used for diseases in which abnormal cells dividewithout control and are able to invade other tissues. There are morethan 100 different types of cancer. Most cancers are named for the organor type of cell in which they start—for example, cancer that begins inthe colon is called colon cancer; cancer that begins in basal cells ofthe skin is called basal cell carcinoma. The main categories of cancerinclude carcinomas, sarcomas, leukemias, lymphomas and myelomas, andcentral nervous system cancers. Some common cancer types include, butare not limited to, bladder cancer, breast cancer, colon and rectalcancer, endometrial cancer, kidney (renal cell) cancer, leukemia, lungcancer, melanoma, non-Hodgkin's lymphoma, pancreatic cancer, prostatecancer, skin cancer (non-melanoma), and thyroid cancer. In oneembodiment, the cancers contemplated for treatment herein include colonand breast cancers.

The terms “comprises”, “comprising”, are intended to have the broadmeaning ascribed to them in U.S. Patent Law and can mean “includes”,“including” and the like.

EMBODIMENTS OF THE INVENTION

In or about January of 2008, largazole was isolated from acyanobacterium of the genus Symploca, and named for its Key Largolocation (Luesch et al, University of Florida). The compounddemonstrates antiproliferative activity in the transformed mammaryepithelial cell line MDA-MB231 with a GI₅₀ of 7.7 nM (Taori et al.2008). In addition, largazole preferentially targets cancer over normalcells, which makes this marine substance an important synthetic targetas well as a potentially valuable cancer chemotherapeutic (Taori et al.2008). The first reported synthesis of largazole was completed by Lueschand co-workers (Ying et al. 2008b), followed by the Phillips group(Nasveschuk et al. 2008), Cramer group (Seiser et al. 2008), Williamsgroup (Bowers et al. 2008), and Ghosh group (Ghosh and Kulkarni 2008).The molecular basis for its anticancer activity has been suggested to beHistone deacetylases (HDAC) inhibition (Ying et al. 2008b).

HDAC inhibitors have been suggested to be a new class of potentanti-cancer agents for the treatment of solid and hematologicalmalignancies. Current inhibitors of HDACs, such as sodium butyrate,Trichostatin A (TSA), suberoylanilide hydroxamic acid (SAHA), FK228, andothers may exhibit their anti-tumor effect by regulating genes and theirprotein products that are required for cell cycle arrest, DNA damagerepair, free radical scavenging and apoptosis (Marks 2010). For example,SAHA has been approved for the treatment of advanced cutaneous T-celllymphoma (Marks 2007). Several other HDAC inhibitors are presently inclinical trials for cancer treatment (Marks 2010).

The structure of largazole comprises a 16-membered macrocycle containinga 4-methylthiazoline fused to a thiazole ring and an octanoic thioesterside chain, a unit rarely found in natural products (Taori et al. 2008;Newkirk et al. 2009). It has been postulated that it is the macrocyclicpart of the compound that interacts with the surface of the HDACprotein, while the side chain would get inserted into HDAC's active siteand chelate zinc, resulting in termination of substrate deacetylation(Newkirk et al. 2009). (FIG. 4).

To further define largazole's pharmacophore, it stands to reason thatupon its entry into the cytoplasm of the cell, the thioester moiety israpidly hydrolyzed to produce the free thiol group, which can nowinteract with the zinc ion at the bottom of the HDAC pocket and potentlyinhibit the enzymatic activity. (FIG. 6).

To validate that largazole thiol is the reactive species, several groupssynthesized a thiol derivative and assessed the biochemical potency intumor cell growth inhibition and in cellular or in vitro HDAC inhibitionassays. These findings indicate that the thiol analogue has similar HDACinhibition using compound-treated cellular extracts (Bowers et al. 2008;Ying et al. 2008a; Ying et al. 2008b). In in vivo experiments, wherecells are treated with largazole or largazole thiol, the parent moleculehas higher potency with respect to HDAC inhibition (IC₅₀ 51 nM vs. 209nM for the thiol metabolite) (Ying et al. 2008a).

With respect to antiproliferative activity, conflicting datasets werepresented by two groups: Ying et al. show that largazole and the thiolanalogue exhibit similar antigrowth activity in HCT116 cells with GI₅₀values 44 and 38 nM, respectively (Ying et al. 2008b). William's grouputilized a series of melanoma cell lines to demonstrate that largazolehas a consistent superior potency (IC₅₀ 45-315 nM) compared to its thiolmetabolite (IC₅₀ 380-2600 nM). They attributed the difference incytotoxicity to the superior permeability of the thioester largazole(Bowers et al. 2008). To measure the deacetylase activity in vitro,purified full length HDAC proteins from class I and class II wereincubated with fluorophore-conjugated substrate and largazole orlargazole thiol. The results not only show that largazole itself is amuch weaker HDAC inhibitor when compared to the reduced version but alsoindicate a pronounced preference of largazole for HDACs 1, 2, and 3 overHDAC6 (Bowers et al. 2008). To account for the lack of difference incellular-base assays, it is possible that the thioester is cleaved underexperimental conditions.

In addition, since hydroxyls do not chelate zinc, a replacement of —SHwith —OH impeded the toxic effect as well as inhibitory activity in HDACassay (Bowers et al. 2008); (Ying et al. 2008a)). Taken together, thethiol is indispensable for both activities; hence one may speculate thatinhibition of HDAC promotes its antitumor effect. From a biosynthesispoint of view, nature produced largazole as a prodrug rather than atarget reactive species to increase its stability and to protect it fromunwanted oxidation (Ying et al. 2008b). Interestingly, an analogousprotect-and-liberate mechanism has been observed in a natural substance,FK228 (Shigematsu et al. 1994), (Ueda et al. 1994a; Ueda et al. 1994b).This distinctive cyclic compound contains a disulfide bond, which uponhydrolysis by glutathione reductase to butenyl thiol extends toward thezinc residue to terminate HDAC's activity. (FIG. 6; and (Furumai et al.2002)).

A series of analogues were prepared to test the optimal length of theoctanoyl chain since it is the linker that gets inserted into the HDACpocket to chelate zinc, which results in attenuation of HDAC biologicalactivity. It is believed that largazole as well as FK228 incorporate afour atom linker between the macrocycle and the zinc binding group. Amacrocycle that lacks the entire octanoyl chain can neither inhibitHDACs nor does it have any toxic activity in cells, which furtherauthenticates the importance of the thiol group in the role of largazoleas an HDAC inhibitor. Neither shortening nor lengthening of thealiphatic chain is an advantageous structural modification as measuredby in vivo and in vitro HDAC assays as well as by cell viability assayagainst the HCT116 colon cancer cell line. (Table 2). These resultssuggest that the natural length of the largazole tail is optimal (Yinget al. 2008a; Ying et al. 2008b; Newkirk et al. 2009). Furthemore, twochanges within the cap region were investigated and reported by Leuschand associates: a substitution of valine to alanine and a largazoleepimer (17R) (Ying et al. 2008a). The Val-, Ala compound showed a 2-folddecrease in all inhibitory activities when compared to largazole,indicating that the valine residue can be easily interchanged. An epimeranalogue behaved poorly as an HDAC inhibitor, alluding to the importanceof the S configuration at position C17 (Ying et al. 2008a). Recently,more structure activity relationship studies on largazole were carriedout by Zeng et al (Zeng et al. 2010), where they replaced valine withleucine and phenylalanine and observed that the inhibitory activityagainst several cancer cell lines was slightly decreased (e.g. GI₅₀ forLargazole was 80 nM while 560 nM and 260 nM was measured for Leu 1 andPhe 1 respectively in HCT 116 cells). Interestingly, when valine wasexchanged for tyrosine, which resulted in lowering the potency againstcancer cells, it greatly increased GI₅₀ for normal cells, exceedinglyimproving the therapeutic window (HCT-116: GI₅₀ 0.39 μM; A549: GI₅₀ 1.46μM) over the normal cell lines (HEK293:GI₅₀ 100 μM; HLF: GI₅₀ 100 μM,while largazole's GI₅₀ in HEK293 is 1.36 μM and 0.98 μM in HLF cells).Hence it was suggested that placing Tyr on largazole could force thecompound to opt for HDACs in cancer instead of normal cells (Zeng et al.2010).

Consequently, macrocyclic HDAC inhibitors such as largazole showpotential as a tool to study the biology of HDACs while at the sametime, due to largazole's preference towards killing cancer cells vs.normal cells, it holds enormous promise as a cancer therapeutic (i.e.,comprises a large therapeutic window). The attractiveness of largazolealso resides in the fact that it is highly selective towards the class Ideacetylases, a feature rarely found in HDAC inhibitors.

In one embodiment, the present invention contemplates a method forimproving upon largarzole's structure-activity relationships by creatinganalogs of largazole and assessing their antiproliferative effects incolon and breast cancer cell lines.

In one embodiment, the present invention provides methods for screeningof compounds of present invention to determine their effect ofinhibition on cancer cell growth.

In another embodiment, the present invention provides methods toincrease largazole's potency and selectivity by creating a 16-membermacrocycle backbone.

In yet another embodiment, the present invention provides novel analogsof largazole by breaking the ring of the 4-methylthiazoline whichresults in improving specificity of antitumor effects, for example an invivo antitumor effect of the novel analogs are realized to a comparableefficacy relative to compounds where the 4-methylthiazoline ring isunbroken yet the number of genes impacted by the novel analog is only ⅓of what has been changed by largazole treatment at 24 hr. Thisobservation suggests that the novel analogs are distinct from largazoleand likely have fewer side effects.

In yet another embodiment, valine of the largazole molecule was replacedwithin the macrocycle with an amino acid selected from the groupconsisting of glycine, alanine, leucine, and isoleucine.

In yet another embodiment, a valine substitution with a glycine improvedthe effectiveness of derivative compound by 3-fold.

In yet another embodiment of the invention, a pharmaceutical compositionis provided comprising, in addition to one or more compounds describedherein, at least one pharmaceutically-acceptable carrier. Thecomposition can take any suitable form for the desired route ofadministration. Where the composition is to be administered orally, anysuitable orally deliverable dosage form can be used, including withoutlimitation tablets, capsules (solid or liquid filled), powders,granules, syrups and other liquids, elixirs, inhalants, troches,lozenges, and solutions. Injectable compositions or i.v. infusions arealso provided in the form of solutions, suspensions, and emulsions.

In yet another embodiment, a pharmaceutical composition according to thepresent invention may contain one or more additional therapeutic agents,for example, to increase the efficacy or decrease side effects. In someembodiments, accordingly, a pharmaceutical composition further containsone or more additional therapeutic agents selected from activeingredients useful to treat or inhibit disease mediated directly orindirectly by HDAC. Examples of such active ingredients are, withoutlimitation, agents to treat or inhibit cancer, Huntington's disease,cystic fibrosis, liver fibrosis, renal fibrosis, pulmonary fibrosis,skin fibrosis, rheumatoid arthritis, diabetes or heart failure.

In yet another embodiment, an additional therapeutic agent to beincluded is an anti-cancer agent. Examples of an anti-cancer agentinclude, but are not limited to, alkylating agents such ascyclophosphamide, dacarbazine, and cisplatin; anti-metabolites such asmethotrexate, mercaptopurine, thioguanine, fluorouracil, and cytarabine;plant alkaloids such as vinblastine, and paclitaxel; antitumorantibiotics such as doxorubicin, bleomycin, and mitomycin;hormones/antihormones such as prednisone, tamoxifen, and flutamide;other types of anticancer agents such as asparaginase, rituximab,trastuzumab, imatinib, retinoic acid and derivatives, colony stimulatingfactors, amifostine, camptothecin, topotecan, thalidomide analogs suchas lenalidomide, CDK inhibitors, proteasome inhibitors such as Velcadeand other HDAC inhibitors.

In yet another embodiment, the present invention provides a method ofinhibiting or treating diseases arising from abnormal cell proliferationand/or differentiation in a subject in need thereof, comprisingadministering to said subject a therapeutically effective amount of oneor more compounds according to the present invention. In one embodiment,the method of inhibiting or treating disease comprises administering toa subject in need thereof, a composition comprising an effective amountof one or more compounds of the invention and a pharmaceuticallyacceptable carrier. The composition to be administered may furthercontain a therapeutic agent such as anti-cancer agent.

While the invention has been particularly shown and described withreference to a number of embodiments, it would be understood by thoseskilled in the art that changes in the form and details may be made tothe various embodiments disclosed herein without departing from thespirit and scope of the invention and that the various embodimentsdisclosed herein are not intended to act as limitations on the scope ofthe claims. All references cited herein are incorporated in theirentirety by reference.

Compounds of the Invention

The compounds of the invention are defined herein by their chemicalstructures and/or chemical names. The compounds of the invention aregenerally named according to the IUPAC or CAS nomenclature system.Abbreviations that are well known to one of ordinary skill in the artmay be used. When a compound is referred to by both a chemical structureand a chemical name, and the chemical structure and chemical nameconflict, the chemical structure is determinative of the compound'sidentity.

Compounds of Formula I of the present invention are synthesizedaccording the generic scheme, Scheme I:

Aryl or Heteroaryl acid intermediates of general Formula 1 and amineintermediates of general Formula 2 can be synthesized by well-knownmethods available in the art or commercially available (Sigma-Aldrich;Advanced Chem Tech; Pep tech; Synthatech). Coupling of acids of Formula1 and amines of Formula 2 provides amides of Formula 3, by knowncoupling methods using suitable reagents such as EDCI(1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) or HATU(2-(7-Aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate). Hydrolysis of the esters of Formula 3 providesacids of Formula 4, which in turn can be coupled with amines of Formula5 to yield compounds of Formula 6. Examples of coupling agents are EDCI(1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) or HATU(2-(7-Aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate). Hydrolysis and removal of the amine protectinggroup of compounds of Formula 6, followed by macrocyclization providemacrolactams of general Formula 7. Macrocylzation can be achieved byreacting the deprotected amino acids of compounds of Formula 6 withdiisopropyethylamine, HATU(2-(7-Aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate) in a solvent such as Tetrahydrofuran. Olefins crossmetathesis reaction of compounds of Formula 7 convert to compounds ofFormula I of the present invention.

EXAMPLES

The following examples are provided for illustrative purposes only andare not intended to limit the scope of the invention.

Example 1S-(E)-4-(7S,10S)-7-isopropyl-4,4-dimethyl-2,5,8,12-tetraoxo-9-oxa-16-thia-3,6,13,18-tetraazabicyclo[13.2.1]octadeca-1(17),15(18)-dien-10-yl)but-3-enyl octanethioate

Step 1: Preparation of methyl2-(2-((tert-butoxycarbonylamino)methyl)thiazole-4-carboxamido)-2-methylpropanoate

To a round bottom flask with 50 mL methylene chloride was added2-((tert-butoxycarbonylamino)methyl)thiazole-4-carboxylic acid (2.0 g,7.74 mmole) and methyl 2-amino-2-methylpropanoate hydrochloride salt(1.25 g, 8.13 mmole). To the mixture, triethylamine (5.4 mL, 38.7 mmole)was then added, followed by1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (2.97 g, 15.5 mmole) andhydroxybenzotriazole (2.09 g, 15.5 mmole). The resulting mixture wasstirred at room temperature overnight. The mixture was then diluted withmethylene chloride. The mixture was then washed with water and theaqueous layer was extracted with methylene chloride. The combinedorganic layer was then dried over Na₂SO₄ and filtered. The filtrate wasconcentrated and purified by silica gel column chromatography elutingwith 1:1 EtOAc/hexane to provide the desired product (2.40 g, 87%yield).

Step 2: Preparation of2-(2-((tert-butoxycarbonylamino)methyl)thiazole-4-carboxamido)-2-methylpropanoicacid

Methyl 2-(2-((tert-butoxycarbonylamino)methyl)thiazole-4-carboxamido)-2-methylpropanoate (2.4 g, 6.7 mmole) wasdissolved in 10 mL methanol and 3 mL water. To the mixture, lithiumhydroxide monohydrate (0.56 g, 13.4 mmole) was then added. The reactionwas stirred at room temperature until TLC indicated complete consumptionof starting material. To the mixture, water and EtOAc were added, andthe aqueous layer was acidified with 2 N HCl to about PH 7. The layerswere separated and the aqueous layer was extracted with EtOAc. Thecombined organic layer was dried over Na₂SO₄ and concentrated to providethe desired product as a white solid (2.3 g, quantitative yield).

Step 3: Preparation of tert-butyl(3S)-3-{[(2S)-2-(2-{[2-({[(tert-butoxy)carbonyl]amino}methyl)-1,3-thiazol-4-yl]formamido}-2-methylpropanamido)-3-methylbutanoyl]oxy}pent-4-enoate

To a solution of 2-(2-((tert-butoxycarbonylamino)methyl)thiazole-4-carboxamido)-2-methylpropanoic acid (1.26 g, 3.67mmole) in 10 mL Dimethylformamide was addedHBTU(O-Benzotriazole-N,N,N′,N′-tetramethyl-uronium-hexafluoro-phosphate)(1.67 g, 4.40 mmole) and diisopropylethylamine (2.0 mL, 11.0 mmole). Theresulting reaction mixture was stirred at room temperature for 10minutes. (S)-tert-butyl 3-((S)-2-amino-3-methylbutanoyloxy)pent-4-enoate(1.0 g, 3.67 mmole) was added, and the resulting mixture was stirredovernight. Water and EtOAc were added and the layers were separated. Theaqueous layer was extracted with EtOAc, the combined organic layer wasdried over Na₂SO₄ and concentrated. It was purified by columnchromatography, eluted with 1:1 Hex/EtOAc to get the desired product(2.1 g, 96% yield).

Step 4: Preparation of(7S,10S)-7-isopropyl-4,4-dimethyl-10-vinyl-9-oxa-16-thia-3,6,13,18-tetraazabicyclo[13.2.1]octadeca-1(17),15(18)-diene-2,5,8,12-tetraone

A solution of(7S,10S)-7-isopropyl-4,4-dimethyl-10-vinyl-9-oxa-16-thia-3,6,13,18-tetraazabicyclo[13.2.1]octadeca-1(17),15(18)-diene-2,5,8,12-tetraone(200 mg, 0.33 mmole) in 10 mL methylene chloride was cooled to 0° C. Tothe mixture, 10 mL trifluoroacetic acid was added. The mixture wasstirred at 0° C. for 1.5 hours. The mixture was concentrated andazeotroped three times with toluene and one time with tetrahydrofuran togive the amine-acid. In a second round bottom flask was placed HATU (381mg, 1.0 mmole) and N,N-diisopropylethylamine (0.51 ml, 2.85 mmole) in 70mL tetrahydrofuran. The mixture was cooled to 0° C. The solution of theabove crude amine-acid in 14 mL tetrhydrofuran was then added over 8hours via a syringe pump. The reaction mixture was then stirredovernight in a cold room at 4° C. It was then warmed up to roomtemperature and stirred for 2 hours. The reaction mixture was thenquenched with water. The layers were separated and the aqueous layer wasextracted with EtOAc. The combined organic layer was dried over Na₂SO₄and concentrated. The mixture was purified by silica gel chromatography,eluted with EtOAc to give the desired product. It was further purifiedby reverse phase chromatography, eluted with 0-100% water/CH₃CN to getthe desired product (45 mg, 32% yield.

Step 5: Preparation of(7S,10S)-10-((E)-4-bromobut-1-enyl)-7-isopropyl-4,4-dimethyl-9-oxa-16-thia-3,6,13,18-tetraazabicyclo[13.2.1]octadeca-1(17),15(18)-diene-2,5,8,12-tetraone

To a mixture of(7S,10S)-7-isopropyl-4,4-dimethyl-10-vinyl-9-oxa-16-thia-3,6,13,18-tetraazabicyclo[13.2.1]octadeca-1(17),15(18)-diene-2,5,8,12-tetraone(20 mg, 0.047 mmole) in 2 mL 1,2-dichloroethane was added4-bromo-1-butene (25.6 mg, 0.19 mmole) and Zhan-1 catalyst (3.2 mg,0.0047 mmole). The mixture was briefly degassed and then heated in asealed tube at 85° C. overnight. The crude product was concentrated andpassed through a silica gel plug to get a mixture of the desired productand recovered starting material (2:1 ratio). It was further purified byreversed phase chromatography, eluted with 0-80% water/CH₃CN to get thedesired product (8 mg, 32% yield).

Step 6: Preparation ofS-(E)-4-((7S,10S)-7-isopropyl-4,4-dimethyl-2,5,8,12-tetraoxo-9-oxa-16-thia-3,6,13,18-tetraazabicyclo[13.2.1]octadeca-1(17),15(18)-dien-10-yl)but-3-enyloctanethioate

To a mixture of(7S,10S)-10-((E)-4-bromobut-1-enyl)-7-isopropyl-4,4-dimethyl-9-oxa-16-thia-3,6,13,18-tetraazabicyclo[13.2.1]octadeca-1(17),15(18)-diene-2,5,8,12-tetraone(15 mg, 0.028 mmole) in 1 mL acetone at room temperature was added K₂CO₃(16 mg, 0.12 mmole) and octanethioic S-acid (14 mg, 0.085 mmole). Themixture was stirred at room temperature for four hours. The solvent wasevaporated. The crude mixture was passed through a silica gel plug. Itwas further purified by reversed phase chromatography, eluted with 0-90%water/CH3CN to get the desired product (4 mg, 23% yield). ¹H-NMR (300MHz, CDCl₃) δ: 8.02 (s, 1H), 7.62 (s, 1H), 6.47 (d, J=10.85 Hz, 1H),6.33 (dd, J=8.65, 4.50 Hz, 1H), 5.83-5.67 (m, 2H), 5.64-5.56 (m, 1H),5.18 (dd, J=17.31, 8.22 Hz, 1H), 4.64 (dd, J=9.87, 3.97 Hz, 1H), 4.37(dd, J=17.21, 4.01 Hz, 1H), 2.87 (t, J=7.2 Hz, 2H), 2.81-2.61 (m, 2H),2.51 (t, J=7.5 Hz, 2H), 2.30 (m, 3H), 1.90 (s, 3H), 1.59 (m, 2H), 1.57(s, 3H), 1.27 (m, 8H), 0.88 (m, 5 H), 0.69 (d, J=6.82 Hz, 3H)

Alternative route for the Preparation ofS-(E)-4-(7S,10S)-7-isopropyl-4,4-dimethyl-2,5,8,12-tetraoxo-9-oxa-16-thia-3,6,13,18-tetraazabicyclo[13.2.1]octadeca-1(17),15(18)-dien-10-yl)but-3-enyloctanethioate

To a mixture of(7S,10S)-7-isopropyl-4,4-dimethyl-10-vinyl-9-oxa-16-thia-3,6,13,18-tetraazabicyclo[13.2.1]octadeca-1(17),15(18)-diene-2,5,8,12-tetraone(32.5 mg, 0.077 mmole, prepared from step 4) and S-but-3-enyloctanethioate (33 mg, 0.15 mmole) in 1 mL dichloroethane was added Grelacatalyst (5 mg, 0.077 mmole). The mixture was purged with Argon for afew minutes and heated to 85° C. for two hours. The additionaloctanethioate (16.5 mg, 0.077 mmole) and Grela catalyst (5 mg, 0.077mmole) were added. It was stirred for two more hours. The solvent wasevaporated and the mixture was purified by silica gel chromatography toget the desired product (21.3 mg, 46%). MS (ESI) [M+Na⁺]⁺=631.3

Example 2S-(E)-4-((7S,10S)-7-isopropyl-4,4-dimethyl-2,5,8,12-tetraoxo-9-oxa-16-thia-3,6,13,18-tetraazabicyclo[13.2.1]octadeca-1(17),15(18)-dien-10-yl)but-3-enyl ethanethioate

To a mixture of(7S,10S)-10-((E)-4-bromobut-1-enyl)-7-isopropyl-4,4-dimethyl-9-oxa-16-thia-3,6,13,18-tetraazabicyclo[13.2.1]octadeca-1(17),15(18)-diene-2,5,8,12-tetraone(20 mg, 0.038 mmole) (prepared as step 5 of Example 1) in 0.5 mL acetoneat room temperature was added K₂CO₃ (10.5 mg, 0.08 mmole) and thioaceticacid (6 mg, 0.08 mmole). The mixture was stirred at room temperature forone hour. The solvent was evaporated. The crude mixture was purified bysilica gel chromatograph, eluted with EtOAc, then 10% MeOH in EtOAc toget the desired product (19 mg, 96% yield). MS (ESI) [M+Na⁺]⁺=547.2

Example 3S-(E)-4-(7S,10S)-7-isopropyl-2,5,8,12-tetraoxo-9-oxa-16-thia-3,6,13,18-tetraazaspiro[bicyclo[13.2.1]octadeca[1(17),15(18)]diene-4,1′-cyclopropane]-10-yl)but-3-enyloctanethioate

This compound was prepared according the procedure of Example 1 usingappropriate starting materials. MS (ESI) [M+Na⁺]⁺=629.2

Example 4S-(E)-4-((7S,10S)-4,4,7-trimethyl-2,5,8,12-tetraoxo-9,16-dioxa-3,6,13,18-tetraazabicyclo[13.2.1]octadeca-1(17),15(18)-dien-10-yl)but-3-enyloctanethioate

This was prepared according the procedure of Example 1 using appropriatestarting materials. MS (ESI) [M+Na⁺]⁺=587.3

Example 5(7S,10S)-4,4-dimethyl-10-[(1E)-4-(octanoylsulfanyl)but-1-en-1-yl]-7-(propan-2-yl)-9-oxa-3,6,13,19-tetraazabicyclo[13.3.1]nonadeca-1(18),15(19),16-triene-2,5,8,12-tetrone

This was prepared according the procedure of Example 1 using appropriatestarting materials. MS (ESI) [M+Na⁺]⁺=625.3

Example 6 Dimer(7S,10S)-7-isopropyl-10-((E)-4-(((E)-4-(7R,10R)-7-isopropyl-4,4-dimethyl-2,5,8,12-tetraoxo-9-oxa-16-thia-3,6,13,18-tetraazabicyclo[13.2.1]octadeca-1(17),15(18)-dien-10-yl)but-3-enyl)disulfanyl)but-1-enyl)-4,4-dimethyl-9-oxa-16-thia-3,6,13,18-tetraazabicyclo[13.2.1]octadeca-1(17),15(18)-diene-2,5,8,12-tetraone

To a solution ofS-(E)-4-(7S,10S)-7-isopropyl-4,4-dimethyl-2,5,8,12-tetraoxo-9-oxa-16-thia-3,6,13,18-tetraazabicyclo[13.2.1]octadeca-1(17),15(18)-dien-10-yl)but-3-enyloctanethioate (10 mg, 0.016 mmole) in 2 mL CH₃CN was added aqueousNH₃(28.9%, 0.2 mL). The mixture was stirred at room temperature for 16h. Then 0.2 mL additional aqueous ammonium was added, and the reactionwas stirred for a day. Additional 0.2 mL of aqueous ammonium was addedand the resulting mixture was stirred overnight. Another 0.1 mL ofaqueous NH₃ was added and stirred for 6 hours. It was concentrated andthe residue was purified by a silica gel column chromatography, elutedwith EtOAc/MeOH (10/1). The fractions that contained the desired productwere combined. It was purified again with reversed phase chromatography,gradient elution with 0-80% CH₃CN/water to get the desired product (6.5mg, 82%). MS (ESI) [M+Na⁺]⁺=985.3

Following are few non limiting examples of compounds of Formula 1 ofScheme I:

Following are few non limiting examples of compounds of Formula 2 ofScheme I:

Example 7 Cell Culture

SW480, HCT116, and MDA-MB231 cell lines were purchased from the AmericanTissue Culture Collection. SW480 and MDA-MB231 cells were grown inDulbecco's modified Eagle's medium supplemented with 10% fetal calfserum at 37° C. in a humidified 5% CO2 atmosphere. The HCT116 cell linewas cultured in McCoy's 5a medium [ATCC; Cat. No. 30-2007] with 1.5 mML-glutamine and 10% fetal bovine serum [ATCC; Cat. No. 30-2020]. HMEcells (Clonetics, San Diego, Calif.; donor 4144) were culturedserum-free in Clonetics' recommended medium and supplements (52 μg/mlbovine pituitary extract, 0.5 μg/ml hydrocortisone, 0.01 μg/ml humanepidermal growth factor, 5 μg/ml insulin, 50 μg/ml gentamicin and 50ng/ml amphotericin-B).

Example 8 Cell Viability Assay

8,000 cells from different cancers were plated in flat-bottomed 96-wellmicroplates. (Background control wells lacking the cells but containingthe same volume of media were included in each assay plate). 24 hoursafter seeding, new media was added. To assess the in vitro cytotoxicity,each compound was dissolved in DMSO and prepared immediately before theexperiments and was diluted into complete medium before addition to cellcultures. Test compounds were then added to the culture medium fordesignated decreasing concentrations (600 nM to 10 nM). Cell viabilitywas determined 48 hours later using crystal violet dye (Sigma-Aldrich),which was solubilized in ethanol, and absorbance was measured at 588 nmusing a Tecan Sail re II plate reader. Experiments were performed inreplicates of six, and concentration-response curves were generated bynonlinear least square regression analysis of the data using GraphPadPrism (San Diego, Calif.). The growth inhibition (GI₅₀) for eachcompound was defined as a concentration of drug leading to a 50%reduction in A588 compared with controls.

Example 9 Western Blotting

For western blot analysis, total protein extracts were prepared bylysing cells in lysis buffer (50 mM Tris-C1 [pH 8.0], 5 mM EDTA, 150 mMNaCl, 1% NP-40, 0.1% SDS, and 1 mM phenylmethylsulfonyl fluoride). 50 μgof total soluble proteins were separated by SDS-PAGE. Proteins weretransferred to nitrocellulose membrane and the membrane was blocked for1 hour with 4% nonfat milk, followed by overnight incubation at 4° C.with primary antibodies against acetylated histone H3 (1:1000, Upstate,#06-599), Ezrin (1:10000, Sigma, E-8897), Glyceraldehyde 3-phosphatedehydrogenase (GAPDH; 1:20000, Santa Cruz, sc-47724), histone H3 (H3;1:1000, Santa Cruz, sc-8654). Membranes were then incubated withperoxidase conjugated secondary antibodies for one hour at roomtemperature. Detection was performed using Super Signal WestDura. Theexpression of Ezrin and GAPDH was used as loading control.

Example 10 Assays to Determine the Effect of Largazole and its Analogson Cancer Cell Growth

8,000 cells from different cancer or nontransformed cells were plated inflat-bottomed 96-well microplates. (Background control wells lacking thecells but containing the same volume of media were included in eachassay plate). 24 hours after seeding, new media was added. To assess thein vitro cytotoxicity, each compound was dissolved in DMSO and preparedimmediately before the experiments and was diluted into complete mediumbefore addition to cell cultures. Test compounds were then added to theculture medium for designated decreasing concentrations (600 nM to 10nM). Cell viability was determined 48 hours later using crystal violetdye (Sigma-Aldrich), which was solubilized in ethanol, and absorbancewas measured at 588 nm using a Tecan Safire II plate reader. Experimentswere performed in replicates of six, and concentration-response curveswere generated by nonlinear least square regression analysis of the datausing GraphPad Prism (San Diego, Calif.). The growth inhibition (GI₅₀)for each compound was defined as a concentration of drug leading to a50% reduction in A₅₈₈ compared with controls.

Example 11 DNA Microarray Studies to Determine the Effect of SAHA,Largazole and its Analogs on Gene Expression Profiles of Cancer Cells

HCT116 cells were seeded in triplicate at approximately 60% confluency.After eight hours, the cells were treated with the vehicle control DMSO(0.01%), SAHA (200 μM), Largazole (20 nM) or Example 1 (20 nM). Cellswere incubated for 6 or 24 hours followed by a wash withphosphate-buffered saline. Total RNA was extracted using a RNeasy MiniRNA extraction kit (QIAGEN Inc., Valencia, Calif.) immediately afterwash. Total RNA concentration was determined using a Lambda 800 UV/VISspectrometer (PerkinElmer, Waltham, Mass.) and processed for labelinghybridization, wash, and scan at University of Colorado-Denver HealthSciences Center. Three GeneChip® Human Gene 1.0 ST (Affymetrix, SantaClara, Calif.) arrays were used for each of the time points, cell types,and treatments for a total of 24 arrays. Expression values files foreach sample were generated using the Robust Multichip Average (RMA)algorithm. Differential expression was determined using the R softwarepackage limma to generate linear models and empirical Bayesianstatistics. Genes were considered differentially expressed if the Pvalue, adjusted for multiple testing using the Benjamini and Hochbergmethod, was <5% and the log-2 fold change was ≦1 or ≧−1. GeneSpring GX(Agilent, Santa Clara, Calif.) software was used for hierarchicalclustering and generating heatmaps.

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While the invention has been particularly shown and described withreference to a number of embodiments, it would be understood by thoseskilled in the art that changes in the form and details may be made tothe various embodiments disclosed herein without departing from thespirit and scope of the invention and that the various embodimentsdisclosed herein are not intended to act as limitations on the scope ofthe claims. All references cited herein are incorporated in theirentirety by reference.

TABLE 1 Zn2+ Class Enzymes Dependent Localization Expression I HDAC1,HDAC2, Yes Nucleus Ubiquitous HDAC3, HDAC8 IIa HDAC4, HDAC5, Yes Nucleusand Tissue HDAC7, HDAC9 cytoplasm specific IIb HDAC6, HDAC10 YesCytoplasm Tissue specific III Sirtuins 1-7 No Variable Variable IVHDAC11 Yes Nucleus and Ubiquitous cytoplasm

TABLE 2 Cell Growth Inhibition GI₅₀ in nM Compound Cell Lines (nM)HCT-116 MDA-MB231 HME Largazole  28 71 ± 8 600 CGN-362 600 600 600CGN-363 600 600 600 CGN-722  9 ± 1 27 ± 6 600 Example 1 16 ± 2  53 600Example 4  17 53 ± 3 600

TABLE 3 Summary of the Number of Genes Whose Expression Levels Changedby Two Fold Upon Treatment with Indicated Chemicals in Comparison toDMSO Control # of Genes 2 Fold-Change Largazole @6 hr 529 Largazole @24hr 566 Example 1@ 6 hr 421 Example 1 @ 24 hr 174 SAHA @ 6 hr 338 SAHA @24 hr 34 TOTAL 32321

1.-19. (canceled)
 20. A compound of Formula (I)

wherein A is aryl or heteroaryl, optionally substituted with one or moregroups selected from C₁-C₁₀ alkyl, C₃-C₁₀ cycloalkyl, C₃-C₁₀heterocycloalkyl, aryl, heteroaryl, halo, hydroxyl, —CN, —COOH, —CF₃,—OCH₂F, —OR₂₀, —NR₂OR₂₂, —NCOR₂OR₂₂ and —CONR₂OR₂₂; Z is —(CH₂)_(n)SR₁₂;R₁ and R₂ are independently H, C₁-C₁₀ alkyl, C₃-C₁₀ cycloalkyl, C₃-C₁₀heterocycloalkyl, or R₁ and R₂ together, or one of the R₁ and R₂together with R₉ form a C₃-C₁₀ cycloalkyl or C₃-C₁₀ heterocycloalkyl,wherein the C₁-C₁₀ alkyl, C₃-C₁₀ cycloalkyl and C₃-C₁₀ heterocycloalkylare optionally substituted with one or more groups selected from C₁-C₁₀alkyl, C₃-C₁₀ cycloalkyl, C₃-C₁₀ heterocycloalkyl, aryl, heteroaryl,halo, hydroxyl, —CN, —COOH, —CF₃, —OCH₂F, —OR₂₀, —NR₂OR₂₂, —NCOR₂OR₂₂and —CONR₂₀R₂₂; R₃ and R₄ are independently H, C₁-C₁₀ alkyl, C₃-C₁₀cycloalkyl, C₃-C₁₀ heterocycloalkyl, or R₃ and R₄ together, or one ofthe R₃ and R₄ together with R₁₀ form a C₃-C₁₀ cycloalkyl or C₃-C₁₀heterocycloalkyl, wherein the C₁-C₁₀ alkyl, C₃-C₁₀ cycloalkyl and C₃-C₁₀heterocycloalkyl are optionally substituted with one or more groupsselected from C₁-C₁₀ alkyl, C₃-C₁₀ cycloalkyl, C₃-C₁₀ heterocycloalkyl,aryl, heteroaryl, halo, hydroxyl, —CN, —COOH, —CF₃, —OCH₂F, —OR₂₀,—NR₂₀R₂₂, —NCOR₂OR₂₂, —CONR₂₀R₂₂ and —S(O)_(m)R₂₀; R₅ and R₆ areindependently H, C₁-C₁₀ alkyl, C₃-C₁₀ cycloalkyl, C₃-C₁₀heterocycloalkyl, or R₅ and R₆ together, or one of the R₅ and R₆together with R₁₁ form a C₃-C₁₀ cycloalkyl or C₃-C₁₀ heterocycloalkyl,wherein the C₁-C₁₀ alkyl, C₃-C₁₀ cycloalkyl and C₃-C₁₀ heterocycloalkylare optionally substituted with one or more groups selected from C₁-C₁₀alkyl, C₃-C₁₀ cycloalkyl, C₃-C₁₀ heterocycloalkyl, aryl, heteroaryl,halo, hydroxyl, —CN, —COOH, —CF₃, —OCH₂F, —OR₂₀, —NR₂OR₂₂, —NCOR₂OR₂₂and —CONR₂₀R₂₂; R₇ and R₈ are independently H, F, C₁-C₁₀ alkyl, C₃-C₁₀cycloalkyl, C₃-C₁₀ heterocycloalkyl, or R₇ and R₈ together form a C₃-C₁₀cycloalkyl or C₃-C₁₀ heterocycloalkyl, wherein the C₁-C₁₀ alkyl, C₃-C₁₀cycloalkyl and C₃-C₁₀ heterocycloalkyl are optionally substituted withone or more groups selected from C₁-C₁₀ alkyl, C₃-C₁₀ cycloalkyl, C₃-C₁₀heterocycloalkyl, aryl, heteroaryl, halo, hydroxyl, —CN, —COOH, —CF₃,—OCH₂F, —OR₂₀, —NR₂OR₂₂, —NCOR₂OR₂₂ and —CONR₂OR₂₂; R₉ is independentlyH, C₁-C₁₀ alkyl, C₃-C₁₀ cycloalkyl, C₃-C₁₀ heterocycloalkyl, or togetherwith one of R₁ and R₂ form a C₃-C₁₀ cycloalkyl or C₃-C₁₀heterocycloalkyl, wherein the C₁-C₁₀ alkyl, C₃-C₁₀ cycloalkyl and C₃-C₁₀heterocycloalkyl are optionally substituted with one or more groupsselected from C₁-C₁₀alkyl, C₃-C₁₀ cycloalkyl, C₃-C₁₀ heterocycloalkyl,aryl, heteroaryl, halo, hydroxyl, —CN, —COOH, —CF₃, —OCH₂F, —OR₂₀,—NR₂OR₂₂, —NCOR₂OR₂₂ and —CONR₂₀R₂₂; R₁₀ is independently H, C₁-C₁₀alkyl, C₃-C₁₀ cycloalkyl, C₃-C₁₀ heterocycloalkyl, or together with oneof R₃ and R₄ form a C₃-C₁₀ cycloalkyl or C₃-C₁₀ heterocycloalkyl,wherein the C₁-C₁₀ alkyl, C₃-C₁₀ cycloalkyl and C₃-C₁₀ heterocycloalkylare optionally substituted with one or more groups selected from C₁-C₁₀alkyl, C₃-C₁₀ cycloalkyl, C₃-C₁₀ heterocycloalkyl, aryl, heteroaryl,halo, hydroxyl, —CN, —COOH, —CF₃, —OCH₂F, —OR₂₀, —NR₂OR₂₂, —NCOR₂OR₂₂and —CONR₂₀R₂₂; R₁₁ is independently H, C₁-C₁₀ alkyl, C₃-C₁₀ cycloalkyl,C₃-C₁₀ heterocycloalkyl, or together with one of R₅ and R₆ form a C₃-C₈cycloalkyl or C₃-C₈ heterocycloalkyl, wherein the C₁-C₁₀alkyl, C₃-C₁₀cycloalkyl and C₃-C₁₀ heterocycloalkyl are optionally substituted withone or more groups selected from C₁-C₈ alkyl, C₃-C₈ cycloalkyl, C₃-C₈heterocycloalkyl, aryl, heteroaryl, halo, hydroxyl, —CN, —COOH, —CF₃,—OCH₂F, —OR₂₀, —NR₂OR₂₂, —NCOR₂OR₂₂ and —CONR₂₀R₂₂; R₁₂ is independentlyH, C₁-C₁₀ alkyl, —COR₂₀, —CONR₂OR₂₂, —OR₂₀, —COOR₂₀, —COCR₂OR₂₂NR₂₀R₂₂,—SR₂₀ or —P(O)(OR₂₄)₂; R₂₀ and R₂₂ are independently H, C₁-C₁₀ alkyl,C₃-C₁₀ cycloalkyl, C₃-C₁₀ heterocycloalkyl, aryl or heteroaryl; R₂₄ isindependently H, C₁-C₁₀ alkyl, C₃-C₁₀ cycloalkyl, C₃-C₁₀heterocycloalkyl, Na, K or Ca; m=1 or 2; and n=1-6; or apharmaceutically acceptable salt thereof.
 21. A compound according toclaim 20, wherein A is a 5-membered heteroaryl ring having at least onenitrogen.
 22. A compound according to claim 20, wherein A is a6-membered heteroaryl ring having at least one nitrogen.
 23. A compoundaccording to claim 21, represented by Formula (II)

wherein R₁-R₁₂, R₂₀, R₂₂, R₂₄, Z, m and n are as defined in claim 20; Land Q are independently S, O, N, or CR₂₆; and R₂₆ is independently H,halo, C₁-C₁₀ alkyl, C₃-C₁₀ cycloalkyl, C₃-C₁₀ heterocycloalkyl, aryl, orheteroaryl, wherein the C₁-C₁₀ alkyl, C₃-C₁₀ cycloalkyl, C₃-C₁₀heterocycloalkyl, aryl and heteroaryl are optionally substituted withone or more groups selected from C₁-C₁₀ alkyl, C₃-C₁₀ cycloalkyl, C₃-C₁₀heterocycloalkyl, aryl, heteroaryl, halo, hydroxyl, —CN, —COOH, —CF₃,—OCH₂F, —OR₂₀, —NR₂OR₂₂, —NCOR₂OR₂₂, and —CONR₂OR₂₂; or apharmaceutically acceptable salt thereof.
 24. A compound according toclaim 21, wherein A is an oxazole or thiazole.
 25. A compound of claim21, wherein R₁ and R₂ are independently H, C₁-C₁₀ alkyl, C₃-C₁₀cycloalkyl, or R₁ and R₂ together form C₃-C₁₀ cycloalkyl; R₃ and R₄ areindependently H, C₁-C₁₀ alkyl, C₃-C₁₀ cycloalkyl, or R₃ and R₄ togetherform C₃-C₁₀ cycloalkyl; R₅ and R₆ are independently H, C₁-C₁₀ alkyl,C₃-C₁₀ cycloalkyl, or R₅ and R₆ together form C₃-C₁₀ cycloalkyl; R₇ andR₈ are independently H, C₁-C₁₀ alkyl, C₃-C₁₀ cycloalkyl, or R₅ and R₆together form C₃-C₁₀ cycloalkyl; R₉, R₁₀ and R₁₁ are independently H orC₁-C₁₀ alkyl; and R₁₂ is —COR₂₀, wherein R₂₀ is independently H orC₁-C₁₀ alkyl.
 26. The compound of claim 20, selected from:S-(E)-4-((7S,10S)-7-isopropyl-4,4-dimethyl-2,5,8,12-tetraoxo-9-oxa-16-thia-3,6,13,18-tetraazabicyclo[13.2.1]octadeca-l(17),15(18)-dien-10-yl)but-3-enyloctanethioate;S-(E)-4-((7S,10S)-7-isopropyl-4,4-dimethyl-2,5,8,12-tetraoxo-9-oxa-16-thia-3,6,13,18-tetraazabicyclo[13.2.1]octadeca-l(17),15(18)-dien-10-yl)but-3-enylethanethioate;S-(E)-4-((7S,10S)-7-isopropyl-2,5,8,12-tetraoxo-9-oxa-16-thia-3,6,13,18-tetraazaspiro[bicyclo[13.2.1]octadeca[l(17),15(18)]diene-4,1T-cyclopropane]-10-yl)but-3-enyloctanethioate; andS-(E)-4-((7S,10S)-4,4,7-trimethyl-2,5,8,12-tetraoxo-9,16-dioxa-3,6,13,18-tetraazabicyclo[13.2.1]octadeca-l(17),15(18)-dien-10-yl)but-3-enyloctanethioate; or a pharmaceutically acceptable salt thereof.
 27. Acompound according to claim 22, represented by Formula (III)

wherein R₁-R₁₂, R₂₀, R₂₂, R₂₄, Z, m and n are as defined in claim 20; L,Q and Y are independently N or CR₂₆; and R₂₆ is independently H, halo,C₁-C₁₀ alkyl, C₃-C₁₀ cycloalkyl, C₃-C₁₀ heterocycloalkyl, aryl, orheteroaryl, wherein the C₁-C₁₀ alkyl, C₃-C₁₀ cycloalkyl, C₃-C₁₀heterocycloalkyl, aryl and heteroaryl are optionally substituted withone or more groups selected from C₁-C₁₀ alkyl, C₃-C₁₀ cycloalkyl, C₃-C₁₀heterocycloalkyl, aryl, heteroaryl, halo, hydroxyl, —CN, —COOH, —CF₃,—OCH₂F, —OR₂₀, —NR₂OR₂₂, —NCOR₂OR₂₂, and —CONR₂OR₂₂; or apharmaceutically acceptable salt thereof.
 28. A compound of claim 27,wherein R₁ and R₂ are independently H, C₁-C₁₀ alkyl, C₃-C₁₀ cycloalkyl,or R₁ and R₂ together form a C₃-C₁₀ cycloalkyl; R₃ and R₄ areindependently H, C₁-C₁₀ alkyl, C₃-C₁₀ cycloalkyl, or R₃ and R₄ togetherform a C₃-C₁₀ cycloalkyl; R₅ and R₆ are independently H, C₁-C₁₀ alkyl,C₃-C₁₀ cycloalkyl, or R₅ and R₆ together form a C₃-C₁₀ cycloalkyl; R₇and R₈ are independently H, C₁-C₁₀ alkyl, C₃-C₁₀ cycloalkyl, or R₇ andR₈ together form a C₃-C₁₀ cycloalkyl; R₉, R₁₀ and R₁₁ are independentlyH or C₁-C₁₀ alkyl; and R₁₂ is —COR₂₀, wherein R₂₀ is independently H orC₁-C₁₀ alkyl.
 29. The compound of claim 20, which is(7S,10S)-4,4-dimethyl-10-[(lE)-4-(octanoylsulfanyl)but-1-en-1-yl]-7-(propan-2-yl)-9-oxa-3,6,13,19-tetraazabicyclo[13.3.1]nonadeca-l(18),15(19),16-triene-2,5,8,12-tetrone;or a pharmaceutically acceptable salt thereof.
 30. The compound ofFormula (IV):

or a pharmaceutically acceptable salt thereof.
 31. A pharmaceuticalcomposition comprising a compound of claim 20 or a pharmaceuticallyacceptable salt thereof and a pharmaceutically acceptable carrier.
 32. Apharmaceutical composition according to claim 31, further comprising oneor more anti-cancer agents.
 33. A method of treating a disease mediatedby a HDAC enzyme, comprising administering to a subject in need thereofa therapeutically effective amount of a compound of claim 20.